Electro-optical devices and methods for hybridization and detection

ABSTRACT

The invention provides an apparatus and method for detection of a target molecule. The apparatus includes a probe labeled with a transition metal-ligand complex that hybridizes with the target to form an initial complex, a metal ion for doping the initial complex and forming a final complex, and a potential means for providing a potential to the final complex to produce a detectable signal indicating the presence of the target after redox reaction. The method of the invention teaches the steps of hybridizing a probe with an attached label to the target to produce an initial complex, adding a metal ion to the initial complex to form a final complex and applying a potential to the final complex to produce a measurable signal.

TECHNICAL FIELD OF THE INVENTION

The invention relates to the field of nucleic acids and moreparticularly to the detection of presence of or a change in sequence orstructure based on hybridized probes.

BACKGROUND OF THE INVENTION

A number of techniques are known and have been developed for detectingthe presence of nucleic acids and their structural changes. However,most of these techniques chemically alter the nucleic acid of interestor change the molecule in some fashion. For instance, modification ofDNA sequences has been accomplished using restriction enzymes. Theseenzymes digest the nucleic acid into defined sequences and alter thedefined order or continuity of the molecule. Restriction enzymes havebeen used in diagnostic medicine and molecular biology as well asgenomics, genotyping, DNA diagnostics, molecular diagnostics and highthroughput screening.

More recently, solutions to hybridization detection use labeled probesand/or targets that provide various optical or electrical signals whenprobes and targets hybridize. This type of technique has significantadvantages over the above-described techniques that require completechemical modification.

Other improved methods that use partial chemical modification are alsoknown in the art. For instance, U.S. Pat. No. 5,591,578 to Meade et al.,teaches the application of ruthenium complexes to the backbone ofnucleic acid molecules. The ruthenium complexes are covalently bound tothe ribose-phosphate backbone of the nucleic acid at predeterminedpositions. In addition, Lee (WO 99/31115 and Biochem. Cell Biol. Vol.71, 1993, 162–168) teaches a number of techniques for hybridizationdetection using the electrical properties of M-DNA. However, each ofthese techniques, as well as others described in the literature, requiremodification of the probe as well as the sample used in the detection.In particular, the method of Lee et al. requires the use of more thanone tag that has been chemically attached to the probe and/or target DNAfor fluorescence detection. The need for the modification of the sampleDNA is a common step for several of the hybridization detectiontechniques. The techniques require extensive sample preparation toobtain and modify DNA that works with these types of tests. Otherdisadvantages with the prior art concern detecting single base pairmismatches in the DNA after hybridization. In order to be able to detectsuch mismatches, very stringent conditions of pH, temperature and saltconcentration must be maintained. (Jonathan A. Prince, Lars, Feuk, W.Mathias Howell, Magnus Jobs, Tesfai Emahazion, Kaj Blennow and AnthonyJ. Brookes, Genome Research, 11, 2001, 152–162) This often leads to higherror rate in the detection of single base mismatches as in singlenucleotide polymorphism (SNP) scoring.

Accordingly, there is an ongoing need for new inventions, methods andtechniques that provide high signal and sensitivity during or after thehybridization process. These techniques should also have broadapplication to high throughput screening and microarray platforms.Furthermore, there is a need for a technique that is simple to use,needs minimal sample preparation and is capable of detecting single basepair mismatches using a simple hybridization procedure.

SUMMARY OF THE INVENTION

The invention provides an apparatus and method for detecting thepresence of a target molecule.

The apparatus comprises a probe labeled with a transition metal-ligandcomplex for hybridizing with the target to form an initial complex,metal ions for doping the initial complex and forming a final complex,and potential means for providing a potential to the final complex toproduce a detectable signal indicating the presence of the target.

The invention also provides a method for detecting the presence of atarget molecule. The method comprises hybridizing a probe having anattached label to a target to produce an initial complex, adding metalions to the initial complex to make a final complex, and applying apotential to the final complex to produce a measurable signal. Inparticular, the method of the invention teaches a technique wherein onlya probe molecule needs to be tagged.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described with reference to thedrawings in which:

FIG. 1 shows a general schematic view of the chemistry of oxidation andreduction chemistry of the electrochemiluminescent compounds used withthe present invention.

FIG. 2 shows a general schematic view of the apparatus of the presentinvention.

FIG. 3(A) shows a general schematic view of the invention with theprobes bound to the electrode surface and the addition of the target tothe probe to produce an initial complex.

FIG. 3(B) shows a general schematic view of the invention after a targethas hybridized to the probe and metal ions have been added to produce afinal complex.

FIG. 3(C) shows a general schematic view when a potential is applied tothe final complex to produce an electrochemiluminescent signal.

FIG. 4 shows the present invention as related to a microarray device.

FIG. 5 shows an enlarged portion of the microarray device shown in FIG.4.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to specific compositions,process steps, or equipment, as such may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a probe” includes more than one probe, reference to “atarget” includes a plurality of targets and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

A “biopolymer” refers to a polymer of one or more types of repeatingunits. Biopolymers are found in biological systems and particularlyinclude peptides and polynucleotides, as well as such compounds composedof or containing amino acid or nucleotide analogs or non-nucleotidegroups. This includes polynucleotides in which the conventional backbonehas been replaced with a non-naturally occurring or synthetic backbone,and nucleic acids in which one or more of the conventional bases havebeen replaced with a synthetic base capable of participating inWatson-Crick type hydrogen bonding interactions. Polynucleotides includesingle or multiple stranded configurations, where one or more of thestrands may or may not be completely aligned with another. While probesand targets of the present invention will typically be single-stranded,this is not essential. Specifically, a “biopolymer” includes DNA(including cDNA), RNA and polynucleotides, regardless of the source.

A “nucleotide” refers to a sub-unit of a nucleic acid and has aphosphate group, a 5-carbon sugar and a nitrogen containing base, aswell as analogs of such sub-units.

An “oligonucleotide” refers to a nucleotide multimer of about 10 to 100nucleotides in length, while a “polynucleotide” includes a nucleotidemultimer having any number of nucleotides.

A “biomonomer” refers to a single unit, which can be linked with thesame or other biomonomers to form a biopolymer (for example, a singleamino acid or nucleotide with two linking groups one or both of whichmay have removable protecting groups). A biomonomer fluid or biopolymerfluid refers to a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

The term “doping” refers to the process of adding at least one metal ionor other conductive molecule or material to a complex, nucleic acid,polymer or biopolymer. The term includes adding the metal ions to anypart or component of the molecules or complexes. The metal ions orconductive molecules or materials need not be added between themolecules themselves, but may contact one or more of the molecules insome manner.

The term “transition metal ligand complex” refers to metal ligandcomplexes having centralized atoms from the transition metals of theperiodic table. Transition metals include and are not limited toruthenium, osmium, iridium etc.

The term “metal ion” refers to divalent and multivalent atoms. Forexample, nickel, zinc, cobalt. etc. Term also should be interpretedbroadly to include elements and complexes from the lanthanide series ofthe periodic table. Metals have characteristic physical properties suchas being efficient in conduction of heat and electricity, malleability,ductibility and a lustrous appearance. Chemically, metals lose electronsto form positive metal ions.

The term “initial complex” refers to a complex that contains at leastone target and labeled probe. The complex may or may not be directlyattached to a surface or substrate.

The term “final complex” refers to a complex that contains at least onemetal ion, target, and labeled probe. The complex may or may not bedirectly attached to a surface or substrate.

The term “potential means” refers to any machine, device, or apparatusfor adding a potential to the final complex. The term is intended to bebroad based and include any and all circuitry whether chemical,electrical or mechanical that will provide a potential to the system andfinal complex. Other means and methods well known in the art areintended to be included in the definition.

The term “aptamer” refers to DNA or RNA molecules that have beenartificially evolved and selected to bind other molecules, viruses, etc.They have many potential uses in medicine and biosciences technology.

The term “derivatives” refers to any molecule that can be produceddirectly from the molecule of interest using synthetic organicchemistry. Derivatives are synthesized molecules that have the originalstructure modified in some way through the addition or deletion offunctional or non-functional groups.

An “array”, unless a contrary intention appears, refers to any one ortwo-dimensional arrangement(s) of addressable regions bearing particularbiopolymer moieties (for example different polynucleotide sequences)associated with that region. An array is “addressable” in that it hasmultiple regions of different moieties (for example, differentsequences) such that a region at a predetermined location (an “address”)on the array (a “feature” of the array) will detect a particular targetor class of targets (although a feature may incidentally detectnon-targets of the feature). In the present case, the polynucleotide (orother) target will be in a mobile phase (typically fluid), while probesfor the target (“probes”) may or may not be mobile (as described in thisapplication). “Hybridizing” and “binding”, with respect topolynucleotides, are used interchangeably. “Binding efficiency” refersto the productivity of a binding reaction, measured as either theabsolute or relative yield of binding product formed under a given setof conditions in a given amount of time. “Hybridization efficiency”refers to a particular sub-class of binding efficiency, and refers tobinding efficiency in the case where the binding components arepolynucleotides. It will also be appreciated that throughout the presentapplication, that words such as “upper”, “lower” are used in a relativesense only. A “set” may have one type of member or multiple differenttypes. “Fluid” as used herein refers to a liquid.

The term “target” refers to a nucleic acid, nucleotide, nucleoside ortheir analogs. The term shall also include nucleotides having modifiedsugars as well as organic and inorganic groups attached to the purine orpyrimidine rings. It shall be broad enough to include reference to RNAor modified RNA in gene expression detection.

The term “probe” refers to a nucleic acid, nucleotide, nucleoside ortheir analogs. The term shall also include nucleotides having modifiedsugars as well as organic and inorganic groups attached to the purine orpyrimidine rings.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, theforegoing description as well as the example that follows are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

FIG. 1 shows a diagram of how the electrochemiluminescence is producedusing the chemistry of the present invention. ML is and abbreviation forthe transition metal-ligand complex. M is an abbreviation for rutheniumor osmium. L stands for one or more ligands such as 2,2′-bipyridine or1,10-phenanthroline. The electrode in the diagram is a working electrodethat obeys standard properties known in the art (Yanbing Zu and Allen J.Bard, Anal. Chem., 72, 2000, 3223–3232). As can be seen in the diagram,the M²⁺L complex initially becomes oxidized at or near the surface ofthe working electrode to form the M³⁺L complex. Similarly, an amine isalso oxidized at the surface of the electrode to form a cation radical.The amine cation radical is then deprotonated to form the carbon radicalof the same compound by losing one proton. The carbon radical compoundthen donates an electron to the M³⁺L complex to form the excited stateof this compound; shown as *M²⁺L. This species then emits energy in theform of light (i.e. electrochemiluminescence is produced).

FIG. 2 shows a general diagram of the apparatus of the presentinvention. The invention provides an apparatus for detecting thepresence of target 5. The apparatus includes a probe 1 labeled with atransition metal-ligand complex 2, a metal ion 3 for doping thehybridized probe 1 and target 5, and potential means 6 for providing apotential to the final complex 10. The linkage between DNA probe andtransition metal-ligand complex can be based on the reactions of aminomodified probes with NHS ester or isothiocyanate activated complexes, orvise versa. It can also be based on the reaction of thiol modifiedprobes and maleimide activated complexes, or vise versa. The followingreferences can be used as an example: (Ewald Terpetschnig, Jonathan D.Dattelbaun, Henryk Szmacinski and Joseph R. Lakowicz, AnalyticalBiochemistry, 251, 1997, 241–245; Gary F. Blackburn, Haresh P. Shah,John H. Kenten, Jonathan Leland, Ralph A. Kamin, John Link, JeffPeterman, Michael J. Powell, Arti Shah, David B. Talley, Surendera K.Tyagi, Elizabeth Wilkins, Tai-Guang Wu and Richard J. Massey, clinicalchemistry, 37(9), 1991, 1534–1539). The final complex 10 produces adetectable signal 7 (not shown in FIG.) from the transition metal-ligandcomplex 2. Probe 1 may be free in solution or attached to surface 8 byany well-known chemistry in the art. For instance, the probes can bedeposited on the solid support via electronic statistic interactions,such as negatively charged DNA probes on a positively charged polylysinesurfaces. The conjugation chemistry mentioned above for covalentlylinking probes and metal-ligand complexes can be applied to thedeposition of probes. Another useful linkage is based on the reaction ofthiol with gold to form Au—S bonds (J. S. Lee, WO 99/31115).Phosphoramidite chemistry may be used for directly attaching the probe 1to the surface 8 (See FIG. 2) by the fast reaction betweenphosporamidite with hydroxyl. This technique is well known in the art.Attachment of the probe 1 may be accomplished by both covalent andnon-covalent bonding methods. (Kazuhito Hashimoto, Masahiro Hiramoto,Tadayoshi Sakata, Hiroji Muraki, Hirofumi Takemura and MasamichiFujihira, The Journal of Physical Chemistry, 91(24), 1987, 6198–6203).

The potential means 6 for providing the potential may include any numberof devices or methods well known in the art. In particular, workingelectrode 13 (shown in FIGS. 2 and 3(A)–(C)) may be an anode or cathodefor oxidizing or reducing electro-active components to the probe. Thispotential produces a detectable signal 7 from the transitionmetal-ligand complex 2 (signal 7 is shown in FIG. 3(C)), when the redoxreaction is completed (details of the circuit or potential means 6 arenot shown in the FIGS.). Potential means 6 may be any apparatus ordevice well known in the art for creating a potential (Mark M. Richterand Karen J. Brewer, Inorganic Chemistry, 32(13), 1993, 2827–2834). Thepotential means 6 may be applied to one or more of the final complexes10. It is also within the scope of the invention that separate potentialmeans 6 be applied to each feature 9 having one or more final complexes10.

FIGS. 3(A)–(C) show the apparatus and method of the present invention.Probe 1 is labeled with a transition metal-ligand complex 2 and isattached to surface 8.

Surface 8 may include any number of materials, surfaces, biopolymers,biomonomers, oligonucleotides, proteins, amino acids chains, aminoacids, aptamers, polynucleotides, co-polymers, or polymers capable ofbonding to, attaching or binding nucleic acids or their derivatives. Forexample, surface 8 may be a microarray surface. In the case that surface8 is a microarray surface, various features 9 can be designated anddefined.

Probe 1 may include a variety of different types of nucleic acids,oligonucleotides, polymers, or aptamers. The important point being thatprobe 1 is designed for capturing or hydridizing to target 5. Probe 1may be of a known or unknown sequence. Probe 1 is attached to surface 8by chemical bonding such as covalent or non-covalent bonding. The probe1 may be attached by any methods of attachment that are well known inthe art.

Transition metal-ligand complex 2 is shown as (ML) in the diagram and isattached at the end of probe 1. Inorganic complex 2 may include anynumber of complexes capable of producing electrochemiluminescence uponoxidation. The ML used in the diagram represents ruthenium and osmiumcomplexes with three 2,2′-bipyridine-like and/or1,10-phenanthroline-like ligands (Yanbing Zu and Allen J. Bard, Anal.Chem., 72, 2000, 3223–3232). Other inorganic complexes may be used withthe invention that are well known in the art. For instance, a number ofinorganic complexes are disclosed in U.S. Pat. Nos. 6,140,138 and6,048,687. The complexes and methods described in these patents as wellas others described supra and infra are hereby incorporated by referenceand may be used with the present invention.

Inorganic complex 2 may also include other metal ions, transition metalsor alloys that are capable of providing chemiluminescent orelectrochemiluminescent signals upon oxidation, reduction or excitation.The transition metal-ligand complex 2 may be attached to probe 1 in avariety of positions or orientations. For example, it may be linked to aDNA backbone ribose or phosphate. It may also be linked to a base ormodified base (U.S. Pat. No. 5,591,578; Gary F. Blackburn, Haresh P.Shah, John H. Kenten, Jonathan Leland, Ralph A. Kamin, John Link, JeffPeterman, Michael J. Powell, Arti Shah, David B. Talley, Surendera K.Tyagi, Elizabeth Wilkins, Tai-Guang Wu and Richard J. Massey, clinicalchemistry, 37(9), 1991, 1534–1539). The diagram shows the label on theend portion of probe 1. This is the most convenient position, because itis easy to label and does not interfere with the bonding between theprobe 1 and target 5.

It should be noted that target 5 may comprise any number of nucleicacids or polymers in various lengths and base pairs. Target 5 may be ofknown or unknown sequence.

FIG. 3(A) shows the addition of target 5 to the initial complex 4. Ifthe target 5 is complementary to the probe 1, the strands hybridize toform the initial complex 4. Hybridization may be complete or partialdepending upon the complementarity of the base pair sequences of target5 and the probe 1.

FIG. 3(B) shows how the final complex 10 is constructed. A metal ion 3is added to the initial complex 4 to form the final complex 10. Themetal ion shown in FIG. 3(B) is in the form of a metal chloride. Metalions with other counter ions may be used as long as the metal salts aresoluble in water and solution can be maintained at a pH of 8.5. Slightlybasic pH is used in order to allow the insertion of one divalent metalion per base pair (Palok Aich, Shaunivan L. Labiuk, Les W. Tari, LouisJ. T. Delbaere, William J. Roesler, Kenneth J. Falk, Ronald P. Steer andJeremy S. Lee, J. Mol. Biol., 294, 1999, 477–485). This allows for theformation of highly conductive DNA. It is known in the art that themetals used in most cases will bond between base pairs in place of onehydrogen bond that would provide Watson-Crick base pairing betweenopposite nucleic acids strands. Metals include and are not limited tozinc, nickel, cobalt etc. The metal ion 3 must be capable of binding tothe final complex 10. Under certain pH conditions, modification of basescan improve overall binding (Palok Aich, Heinz-Bernhard Kraatz andJeremy S. Lee, Journal of Biomolecular Structure & Dynamics, Coversation11, Issue #2, 297–301, Proceedings of the Eleventh Conversation,University of Albany, SUNY, Jun. 15–19, 1999; Personal Discussions.).For instance, this can be accomplished by replacing thymine with5-fluorouricil in the probe and/or target.

FIG. 3(C) shows that upon the addition of an organic amine, the finalcomplex 10 can then be used for detection of the target 5 byelectrochemiluminescence. For instance, a potential is applied to thefinal complex 10 using the working electrode 13. The current is carriedup the probe 1 and the target 5 after the metal ions have been insertedinto the hybridized DNA to form a conductive wire. It should be notedthat the transition metal-ligand complex 2 gets oxidized. As shown inFIG. 1, the application of the potential to the transition metal-ligandcomplex 2 results in electrochemiluminescence in the presence of theorganic amine.

FIGS. 4–5 show the present invention and how it relates to a microarraydevice 15. The microarray device 15 employs a first surface 17 onsubstrate 19 and separated by areas 14. Each microarray device 15contains one or more features 9 used for detection of a particulartarget 5. Each feature 9 contains an apparatus as shown in FIG. 2 withone or more final complexes 10. Generally, a plurality of finalcomplexes 10 will be present for each feature 9. This is done toincrease the signal to noise ratio in the sample (i.e., a strongerelectrochemiluminescent signal 7 will be provided). The invention can,therefore, be used for other applications such as gene expression andsingle nucleotide polymorphism (SNP) detection and mismatch detection.

Having generally discussed the present apparatus and method, adescription of the method of the present invention and how it providesimproved signaling and sequence mismatch detection is in order. Asdescribed, the method of the present invention operates by firsthybridizing probe 1 with attached transition metal-ligand complex 2, totarget 5 to form initial complex 4. Next, the initial complex 4 is madeconductive by adding a metal ion 3 such as zinc, cobalt or nickel.Electrochemiluminescence is obtained by applying a potential to thefinal complex 10. The invention and method are particularly useful inthat they effectively determine whether or not hybridization has takenplace between probe 1 and the target 5, or one or more base pairmismatches exist. The highest signal can be obtained if there is aperfect match between probe 1 and target 5. It should be noted thatdivalent metal ions can not be inserted into the base pair(s) if thebase pairs don't match. The conductivity of such M-DNA wire decreasesdramatically. This will affect the oxidation rate of the final complex10 and lowers the electrochemiluminescence signal. The magnitude of thesignal can be used for determining the quantity and extent of binding orhybridization efficiency between the probe 1 and the target 5. Themethod thus can be applied to single mismatch/multi-mismatch detection.

EXAMPLE 1 Measurement and Detection

In order to obtain detection of the target, the following procedure willbe employed. Target detection is accomplished in the final step afterthe target and the probe have hybridized. These steps have already beendiscussed in some detail. The method and apparatus for this detectionwill be briefly discussed here. A working electrode will be employed. Inparticular, the working electrode will be a gold electrode. A goldelectrode is most effective for making the appropriate measurements forthese types of experiments. In addition, gold electrodes are fairlyeffective reducing agents. However, this example should not beinterpreted to limit the electrode to just gold. Other electrodessimilar to gold may also be employed. Also, electrodes may compriseother materials and surface modification with carboxyl, amino, thiol andhydroxyl groups that are capable of reacting. The functional groupscould be used to covalently link probes to the electrodes or othersurface. The techniques are based on chemical reactions well known inthe art of conjugation chemistry.

EXAMPLE 2 Attachment of Transition Metal-Ligand Complex to Probes

A number of methods known in the art may be used for attachment of thetransition metal-ligand complexes to nucleic acids. The nucleic acidsused in the experiment will be DNA. Other nucleic acids andoligonucleotides can be used. The DNA used as the probe in thisexperiment will be obtained from Promega, Inc. Probes that will be usedwill range in size from 1–100 oligonucleotides.

Initial targets will be produced using polymerase chain reaction of theknown probe sequences. The cDNA or targets will be produced using anAmplicon oligonucleotide synthesizer following standard reactionsequences and procedures. cDNA will be purified from the reaction mixusing a C13 reverse phase HPLC column. As discussed above, initialexperiments will be conducted with known probe and target sequences toensure hybridization. Further experiments will be conducted with genomicDNA digested with restriction endonucleases. In these experiments theprobes will be of a known sequence that identifies a particular gene.Targets need not be of known size or sequence in these furtherexperiments.

Attachment of the transition metal-ligand complexes to the probes shouldbe straight forward. The example discussed below is one effective methodof attaching the transition metal-ligand complexes to the probes. Othermethods known and used in the art may also be employed. A terminalmodified nucleic acid with a thiol functional group will be created onone end of the nucleic acid. The other end of the nucleic acid willcontain an amino acid or amino functional group. The amino group will bereacted with NHS ester or isothiocyanate of a ruthenium complex at aboutpH 8.2–9.3. For more information on the steps and process see (EwaldTerpetschnig, Jonathan D. Dattelbaun, Henryk Szmacinski and Joseph R.Lakowicz, Analytical Biochemistry, 251, 1997, 241–245; Gary F.Blackburn, Haresh P. Shah, John H. Kenten, Jonathan Leland, Ralph A.Kamin, John Link, Jeff Peterman, Michael J. Powell, Arti Shah, David B.Talley, Surendera K. Tyagi, Elizabeth Wilkins, Tai-Guang Wu and RichardJ. Massey, clinical chemistry, 37(9), 1991, 1534–1539). It should benoted that the actual preparation of the product can be produced eitheron solid phase or in solution. The desired product will then be obtainedusing a reverse phase HPLC column. Final products will be storedovernight in a −20° C. freezer.

EXAMPLE 3 Attachment of Probes to Substrate

The thiol terminated probes generated in example 2 above will bedissolved in an alcohol solution containing a gold electrode. Thereaction will be allowed to proceed at ambient conditions for at least acouple of hours and probes will be deposited through Au—S bondformation.

EXAMPLE 4 Hybridization of Probes to Target

Although a number of conditions can be used for effectively hybridizingnucleic acids, the following method should prove most effective with thepresent invention. The hybridization of probe with target will becarried out at 20 mM Tris-HCl buffer at pH 7.5 at 37° C. in the presenceof 10 mM NaCl for several hours. Incubation time and temperature willvary based on the length and composition of the probes.

EXAMPLE 5 M-DNA/Nucleic Acid Formation

The addition of the metal to the hybridized probe and target will beaccomplished according to the following procedure. The formation ofM-DNA will be carried out in CHES buffer at pH=9.0 in the presence of0.1–0.2 mM MCl₂ (M=Zn, Co, Ni etc.). The reaction will be incubated forseveral hours depending on the probe length and composition. Additionalinformation on methods and running conditions can be obtained from(Palok Aich, Shaunivan L. Labiuk, Les W. Tari, Louis J. T. Delbaere,William J. Roesler, Kenneth J. Falk, Ronald P. Steer and Jeremy S. Lee,J. Mol. Biol., 294, 1999, 477–485).

EXAMPLE 6 Electrochemiluminescence

Electrochemiluminescence (ECL) will be accomplished using the procedurediscussed below. After hybridization and formation of the M-DNA, apotential will be applied to oxidize the ruthenium complexes in thepresence of the organic amine. A number of organic amines well known inthe art may be used. In particular, the tertiary amines are effective inproducing ECL in most aqueous buffer systems. The ECL signal can bedetected adjacent to the working electrode surface by a CMOS camera forarray format or fluorimeters. It is expected that perfect matchsequences will produce the highest signal for current orchemiluminescence detection due to the highest conductivity. Presence ofmismatch base pairs should generate a reduced signal due to increase inresistance.

All patents and publications mentioned herein, both supra and infra, arehereby incorporated by reference.

1. A kit for detecting the presence of a target, comprising: a nucleicacid probe that hybridizes to a target to form a first complex, thenucleic acid probe comprising a nucleic acid labeled with a transitionmetal ligand complex, and a free metal ion that binds to the firstcomplex to form an electrically conductive second complex.
 2. The kit ofclaim 1, wherein the second complex produces a signal when an electricpotential is applied.
 3. The kit of claim 1, wherein the metal ion is adivalent cation.
 4. The kit of claim 3, wherein the metal ion is zinc,nickel or cobalt.
 5. The kit of claim 3, wherein the metal ion isinserted between two bases of said complex.
 6. The kit of claim 1,wherein the nucleic acid probe is bound to a solid support.
 7. The kitof claim 6, wherein the solid support is an array.
 8. The kit of claim1, wherein the transition metal ligand complex at a terminus of thenucleic acid.
 9. The kit of claim 1, wherein the transition metal ligandcomplex comprises ruthenium, osmium or derivatives thereof.
 10. A systemfor detecting the presence of a target, comprising: a nucleic acid probethat hybridizes to a target to form a first complex, the nucleic acidprobe comprising a nucleic acid labeled with a transition metal ligandcomplex; a free metal ion that binds to the first complex to form asecond complex; and an electrode for providing a potential to saidsecond complex to produce a detectable signal indicating the presence ofthe target.
 11. The system of claim 10, wherein said signal is anelectrochemiluminescent signal.
 12. A nucleic acid complex having basepairs comprising: a nucleic acid probe comprising a nucleic acid labeledwith a transition metal ligand complex; a target that is hybridized tosaid nucleic acid probe to form a complex; and a free metal ion that isinserted between base pairs of said complex.
 13. A kit for detecting thepresence of a target, comprising: a nucleic acid probe that hybridizesto a target to form a first complex, wherein the nucleic acid probe isattached to a substrate and comprises a nucleic acid labeled with atransition metal ligand complex, and a free metal ion that binds to thefirst complex to form an electrically conductive second complex.
 14. Asystem for detecting the presence of a target, comprising: a nucleicacid probe that hybridizes to a target to form a first complex, whereinthe nucleic acid probe is attached to a substrate and comprises anucleic acid labeled with a transition metal ligand complex; a freemetal ion that binds to the first complex to form a second complex; andan electrode for providing a potential to said second complex to producea detectable signal indicating the presence of the target.
 15. A nucleicacid complex having base pairs comprising: a nucleic acid probecomprising a nucleic acid labeled with a transition metal ligandcomplex, wherein the nucleic acid probe is attached to a substrate; atarget that is hybridized to said nucleic acid probe to form a complex;and a free metal ion that is inserted between base pairs of saidcomplex.